Polymerization of aromatic monomers using derivatives of hematin

ABSTRACT

Hematin, a hydroxyferriprotoporphyrin, is derivatized with one or more non-proteinaceous amphipathic groups. The derivatized hematin can serve as a mimic of horseradish peroxidase in polymerizing aromatic monomers, such as aromatic compounds. These derivatized hematins can also be used as catalysts in polymerizing aromatic monomers, and can exhibit significantly greater catalytic activity than underivatized hematin in acidic solutions. In one embodiment, polymerization is in the presence of a template, along which aromatic monomers align. An assembled hematin includes alternating layers of hematin and a polyelectrolyte, which are deposited on an electrically charged substrate. Assembled hematin can also be used to polymerize aromatic monomers.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/994,998, filed Nov. 27, 2001, which claims the benefit of U.S.Provisional Application No. 60/253,109, filed on Nov. 27, 2000. Theentire teachings of the above application are incorporated herein byreference.

GOVERNMENT SUPPORT

[0002] The invention was supported, in whole or in part, by a grant ONRN0014-00-1-0718 from the Office of Naval Research and a grant DAAD16-01-C-0011 from the U.S. Army Research Office. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Recently, there has been an increased interest in tailoreddevelopment of polyaromatic polymers, particularly polyaromatic polymersthat are electrically conductive and/or have interesting and usefuloptical properties. Examples of electrically conductive polymers includecertain polyanilines, polythiophenes, polypyrroles, and polyphenols.These conductive polyaromatic polymers may be used in a variety ofelectronic devices, including electro-chromic devices, light-emittingdiodes, electrostatic discharge protection, and light weight batteries.Of these polyaromatic polymers, polyanilines are the most extensivelystudied, due largely to superior electrical properties such as highdischarge capacity.

[0004] In addition to the above-named electrical properties, the thermaland structural properties of polyphenols have long been exploited. Inparticular, phenol-formaldehyde resins such as novolacs and resols havefound wide application as wood composites, laminates, foundry resins,abrasives, friction and molding materials, coatings and adhesives, fiberbinders, and flame retardants. The use of formaldehyde in polyphenolsynthesis, however, presents a significant toxicological andenvironmental hazard.

[0005] Despite the industrial utility of polyaromatic polymers, theirsynthesis remains problematic. Known difficulties in the synthesis ofthese polymers include inconsistent product composition, due in part toextensive branching of the polymers. In addition, many of thepolyaromatic polymers are insoluble or sparingly soluble in commonsolvents, leading to poor processability. The use of toxic reagents, asnoted above, is another undesirable feature of current syntheticmethods. A search for new methods of synthesizing polyaromatic polymershas not yet yielded a commercially viable approach.

[0006] Many of the synthetic approaches to forming polyaromatic polymersuse a heme-containing enzyme to catalyze the polymerization. Any suchcatalyst must necessarily be stable and active under acidic conditions,as acidic conditions are required in order to synthesize an electricallyconductive form of a polyaromatic polymer such as polyaniline. Anexample of an enzyme extensively studied for aromatic moleculepolymerization is horseradish peroxidase. Unfortunately, horseradishperoxidase and other peroxidases are inactive at low pH and areprohibitively expensive to use commercially. Hematin has been used tomimic the catalytic activity of horseradish peroxidase. However, despiteits lower cost, hematin is a non-ideal catalyst for commercialpolymerizations because of its low solubility in acidic, aqueous media.The low solubility of hematin under these conditions leads to a low rateof polymerization and poor yields. Therefore, a need exists to develop alow cost, high efficiency means of synthesizing polyaromatic polymers,which is compatible with conditions required to synthesize polymers withcommercially desirable properties.

SUMMARY OF THE INVENTION

[0007] The invention generally is directed to a derivatized hematin; amethod for polymerizing an aromatic monomer with an assembled hematin ora derivatized hematin; and to methods of forming the assembled andderivatized hematins.

[0008] In one embodiment, the invention includes hematin derivatizedwith one or more non-proteinaceous amphipathic groups. In a preferredembodiment, the amphipathic group is polyethylene glycol.

[0009] In another embodiment, the invention includes a method ofpolymerizing aromatic monomers such as anilines or phenols. In apreferred embodiment, the polymerization takes place in the presence ofa template. Typically, the template is anionic.

[0010] In another embodiment, the invention includes a method forpreparing a derivatized hematin, by reacting hematin with an amphipathiccompound. In a preferred embodiment, the hematin is derivatized with anamphipathic compound in the presence of a carboxylic acid activatingcompound and an aprotic base.

[0011] In yet another embodiment, the invention includes an assembledhematin, which includes alternating layers of hematin and apolyelectrolyte on an electrically charged substrate. Preferably, thepolyelectrolyte is cationic.

[0012] In another embodiment, the invention includes a method ofpolymerizing aromatic monomers by contacting an aromatic monomer and atemplate with the assembled hematin. In a preferred embodiment, thearomatic monomer is an aniline or a phenol.

[0013] In another embodiment, the invention includes a method of formingassembled hematin, by alternately depositing one or more layers ofhematin and one or more layers of a polyelectrolyte on an electricallycharged substrate.

[0014] Advantages of the present invention include resolving the currentlimitations of catalysts used in the commercial synthesis ofpolyaromatic polymers, by reducing the cost of the catalyst and byproviding a catalyst that is active and stable over a wide range of pHs.The derivatized hematins of the present invention are also water-solubleand recyclable, virtually eliminating the need for toxic reagents andsolvents, and thus creating an environmentally friendly synthesis forpolyaromatic polymers. In addition, the derivatized hematins of thepresent invention, in a combination with a template, reduce the amountof branching during polymerization, leading to a structurally moreconsistent product.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the functionalization of hematin with polyethyleneglycol (PEG) in the presence of N,N′-carbonyl diimidazole,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and dimethylformamide (DMF).

[0016]FIG. 2 shows the FTIR spectra of hematin and PEG-hematin. Theinset shows an expanded region between 1500 and 1700 cm⁻¹.

[0017]FIG. 3a shows the ¹H NMR spectra of hematin and PEG-hematin inDMF-d₇. The inset shows the disappearance of the hematin carboxylic acidpeak when it is derivatized with PEG.

[0018]FIG. 3b shows the ¹H NMR spectra of hematin and PEG-hematin inD₂0.

[0019]FIG. 4 shows the catalytic activity of hematin and PEG-hematin forthe oxidation of pyrogallol at pH 4.0.

[0020]FIG. 5 shows the UV-vis absorption spectrum of aniline monomersand of polyaniline formed during PEG-hematin catalyzed polymerization.

[0021]FIG. 6 shows the time dependent UV-vis absorption spectra of thepolyaniline-sodium polystyrene sulfonate (SPS) complex formed at pH 4over 2 hours after initiation of polymerization.

[0022]FIG. 7 shows the pH-dependent UV-vis absorption spectra of thepolyaniline-SPS complex formed after initiation of polymerization.

[0023]FIG. 8 shows the UV-vis absorption spectra of a polyaniline-SPScomplex as it is titrated with 1 N NaOH and 1 N HCl, demonstrating thatthe complex can be reversibly dedoped and redoped using base or acid,respectively.

[0024]FIG. 9 shows a cyclic voltammogram of a solution cast film ofpolyaniline-SPS complex synthesized at pH 1.0.

[0025]FIG. 10 shows the pH-dependent UV-vis absorption spectra ofpolyaniline-lignin sulfonate complexes formed during polymerization.

[0026]FIG. 11 shows UV-vis absorption spectra of polyaniline-DNA formedduring PEG-hematin catalyzed polymerization.

[0027]FIG. 12 shows CD spectra of polyaniline-DNA formed duringPEG-hematin catalyzed polymerization.

[0028]FIG. 13 shows time-dependent UV-vis absorption spectra of thepolymerization of 2-methoxy-5-methylaniline catalyzed by PEG-hematin.

[0029]FIG. 14 shows pH-dependent UV-vis absorption spectra ofpolyaniline-dodecylbenzenesulfonic acid complexes formed duringpolymerization.

[0030]FIG. 15 shows UV-vis absorption spectra of a SPS-polyphenolcomplex formed during polymerization.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention generally includes a derivatized hematinand an assembled hematin, along with methods of preparing the hematins.The invention also includes methods of polymerizing aromatic monomers ina reaction catalyzed by an assembled hematin or a derivatized hematin.

[0032] The present invention includes hematin, ahydroxyferriprotoporphyrin, which has been derivatized with one or morenon-proteinaceous amphipathic groups. Examples of amphipathic groupsinclude phosphoglycerides; sphingomyelin; glycolipids; substituted orunsubstituted polyethers and polyalkylene glycols; substituted orunsubstituted polyamines such as polyethyleneimine, polyallylamine, andpoly(diallylamine); polyammonium groups, such as poly(allylammoniumsalts), poly(trimethylallylammonium salts), poly(triethylallylammoniumsalts), poly(dimethyldiallylammonium salts), poly(diethyldiallylammoniumsalts); and polysaccharides such as hydroxypropyl cellulose,hydroxymethyl cellulose, and hydroxyethyl cellulose.

[0033] Preferred amphipathic groups include polyalkylene glycols such aspolyethylene glycol and polypropylene glycol. Preferably, polyethyleneglycol groups have a molecular weight of about 400 to about 100,000, ormore preferably, a molecular weight of about 5,000 to about 15,000.

[0034] In another embodiment, the hematin derivatized with anamphipathic group is soluble over a pH range from about pH 1 to about pH12.

[0035] In another embodiment, the present invention includes a method ofpolymerizing an aromatic monomer, which includes combining the aromaticmonomer with a derivatized hematin catalyst. In a preferred embodiment,the hematin is derivatized with polyethylene glycol. In anotherpreferred embodiment, the derivatized hematin catalyst and the aromaticmonomer are additionally combined with a peroxide to initiate thereaction.

[0036] Aromatic monomers include substituted or unsubstituted aromaticcompounds. Suitable aromatic compounds include4-β-hydroxyphenylazo)pyridine and 4-(p-hydroxyphenylazo)pyridiniummethiodide. Preferred aromatic compounds for polymerization includeaniline, phenol, and 2-methoxy-5-methylaniline.

[0037] Suitable substituents on aromatic monomers will not significantlyreduce the rate of polymerization as compared to an unsubstitutedaromatic monomer (e.g., will not reduce the rate of polymerization bymore than ten-fold). Examples of suitable substituents for aromaticmonomers include, for example, halogen (—Br, —Cl, —I, and —F), —OR, —CN,—NO₂, —COOR, —CONRR₁, —SO_(k)R (where k is 0, 1, or 2), —NRR₁, —SR,haloalkyl groups, and —NH—C(═NH)—NH₂. R and R₁ are independently, —H, analiphatic group, an aralkyl group, a heteroaralkyl group, an aromaticgroup, or a substituted aromatic group. A substituted aromatic monomercan have more than one substituent.

[0038] In a preferred embodiment of the present invention, a template iscombined with the derivatized hematin, an aromatic monomer, and aperoxide, such that the aromatic monomer aligns along the template andpolymerizes to form a complex including the polymerized aromatic monomerand the template. A “template,” as that term is employed herein, isdefined as a polymer or oligomer that can bind, such as ionically bind,to the aromatic monomer being polymerized according to the method of theinvention.

[0039] Suitable template polymers include polyelectrolytes such as ananionic polymer or a cationic polymer. Anionic polymer templates includepolymers that include pendant acid functional groups such aspoly(vinylbenzoic acid) and salts thereof, poly(vinyl polyphosphonicacid) and salts thereof, poly(glutamic acid) and salts thereof,poly(aspartic acid) and salts thereof, poly(acrylic acid), andpoly(maleic acid co-olefin) and salts thereof. Co-olefins that can bepolymerized with maleic acid to form poly(maleic acid co-olefin) include1-propene, 1-butene, 1-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene,and 1-decene. Preferred anionic polymer templates include poly(styrenesulfonic acid) and salts thereof, lignin sulfonic acid and saltsthereof, and dodecylbenzene sulfonic acid and salts thereof.

[0040] Optically active templates can also be employed in thepolymerization method of the invention. When an optically activetemplate is employed, the template can induce macro-asymmetry in thepolymerized aromatic monomer due to the close association of thetemplate with the polymerized aromatic monomer in the complex. Examplesof optically active templates include polynucleic acids and saltsthereof, such as ribonucleic acids and 2′-deoxyribonucleic acids. Othersuitable templates include biological receptors, peptides, proteins,zeolites, caged compounds, phenol red, azo compounds, azo polymers, anddendrimers.

[0041] In a preferred embodiment, the complex of a polymerized aromaticmonomer and a template is a water-soluble complex of a polyaniline and atemplate. Even more preferably, the polyaniline is of theelectrically-conducting emeraldine salt form. Emeraldine is anelectrically-conducting form of polyaniline, and has a characteristicgreen color when protonated, or doped.

[0042] In another preferred embodiment, the complex including apolymerized aromatic monomer and a template is a water-soluble complexof a polyphenol and a template.

[0043] In yet another preferred embodiment, a polymerized aromaticmonomer complexed to an optically active template has a macro-asymmetry.

[0044] A complex of a polymerized aromatic monomer and a template can beprepared by contacting an aromatic monomer, such as an aniline or aphenol, and a template with a derivatized hematin in a solution having apH from about 0 to about 12. Preferably, the solution is buffered, andthe pH ranges from about 0 to about 7, and more preferably ranges fromabout pH 0 to about pH 4. The ratio of aromatic monomer to template(measured as the concentration of template repeat units) can vary from5:1 to 1:5 (aromatic monomer: template repeat unit), and is preferablyfrom about 2:1 to about 1:2, and is even more preferably about 1:1. Acatalytic amount of the derivatized hematin can be added to the reactionmixture either before or after addition of the aromatic monomer. Acatalytic amount of the derivatized hematin is typically between aboutone unit/mL and five units/mL, where one unit will form 1.0 mgpurpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20° C.Preferably, the derivatized hematin is added to the solution afteraddition of the template and aromatic monomer. In a preferredembodiment, a peroxide is also added to the reaction mixture. Theperoxide is added incrementally, such as not to de-activate thederivatized hematin catalyst, until an amount approximatelystoichiometric with the amount of aromatic monomer has been added. Thereaction can be monitored spectroscopically.

[0045] The above polymerization can be carried out in polar solventssuch as ethanol, methanol, isopropanol, dimethylformamide, dioxane,acetonitrile, and diethyl ether, but is preferably carried out in water.

[0046] In one embodiment, the present invention is a method ofderivatizing hematin, which includes reacting hematin with one or moreamphipathic compounds, thereby forming a derivatized hematin. In apreferred embodiment, the hematin is reacted with one or moreamphipathic compounds in the presence of a carboxylic acid activatingcompound and an aprotic base. In a more preferable embodiment, thecarboxylic acid activating compound is a dialkylcarbodiimide. In anotherpreferred embodiment, the amphipathic compound is a substituted orunsubstituted polyalkylene glycol. Even more preferably, thepolyalkylene glycol is polyethylene glycol.

[0047] “Carboxylic acid activating compounds,” as used in the presentinvention, are compounds that serve to couple a nucleophile, such as ahydroxyl, amine, or thiol group, to a carboxylic acid, thereby formingan ester, an amide, or a thioester linkage. Suitable carboxylic acidactivating compounds include dialkylcarbodiimides, preferablydiisopropylcarbodiimide and dicyclohexylcarbodiimide;N,N′-carbonyldiimidazole; nitrophenol, preferably o-nitrophenol andp-nitrophenol; pentahalophenol, preferably pentachlorophenol, andpentabromophenol; N-hydroxysuccinimide; tosyl chloride;1-hydroxybenzotriazole; andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide.

[0048] “Aprotic bases,” as used in the present invention, include baseswithout an exchangeable proton. Suitable aprotic bases includetrialkylamines, such as trimethylamine, triethylamine,diisopropylethylamine and triphenylamine; pyridine; pyrimidine;1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); and 1,3,5-triazine.

[0049] Derivatized hematins of the present invention can be prepared,for example, by reacting about one-half to about ten mole equivalents ofan amphipathic compound, such as polyethylene glycol, with hematin inthe presence of an excess of a carboxylic acid activating compound, andan aprotic base, in an aprotic solvent such as dimethylformamide or anether. The mixture is allowed to stir for about 6 hours to about 6 days,and is then quenched with a large volume of water or other proticsolvent. The unreacted reagents are removed by extraction of thereaction mixture with an organic solvent such as ethyl acetate. Thewater layer is concentrated, preferably by lyophilization, to yield thederivatized hematin.

[0050] In another embodiment, the present invention is assembledhematin, which includes one or more layers of hematin alternating withone or more layers of a polyelectrolyte deposited on a substrate. In apreferred embodiment, the polyelectrolyte is a cationic polymer, such asa poly(dialkyldiallylammonium salt) or a poly(trialkylallylammoniumsalt). More preferably, the polyelectrolyte ispoly(dimethyldiallylammonium chloride).

[0051] In another embodiment, the present invention includes a method ofa polymerizing an aromatic monomer to form a complex of a polymerizedaromatic monomer and a template, by contacting the aromatic monomer andthe template with the assembled hematin. Preferably, the template is ananionic polymer, such as poly(styrene sulfonic acid) or a salt thereof.In another preferred embodiment, the aromatic monomer is a substitutedor unsubstituted aromatic compound, such as an aniline or a phenol. Inyet another preferred embodiment, the complex of the polymerizedaromatic monomer and the template forms in solution or the complex formson the assembled hematin. The complex forming on the assembled hematincan contact one or more layers of hematin or the polyelectrolyte.

[0052] In another embodiment, the present invention includes a method offorming assembled hematin, by alternately depositing layers of hematinand a polyelectrolyte onto an electrically charged substrate.Preferably, the polyelectrolyte is a cationic polymer, and morepreferably is a poly(dialkyldiallylammonium salt) or a(trialkylallylammonium salt), such as poly(dimethyldiallylammoniumchloride).

[0053] Assembled hematins of the present invention can be prepared, forexample, by dipping a charged substrate, such as a negatively-chargedhydrophilized glass slide, into about 0.1 mM to about 100 mM hematinhaving a pH from about 6 to about 12 at about 0° C. to about 50° C. forabout 1 minute to about 100 minutes. The substrate is washed withdeionized water and dried with a stream of gas, such as nitrogen orargon. The substrate with a single layer of hematin is dipped into about0.1 mM to about 100 mM polyelectrolyte having a pH from about 6 to about12 at about 0° C. to about 50° C. for about 1 minute to about 100minutes. The substrate is washed with deionized water and dried with astream of gas, such as nitrogen or argon. The process can then berepeated, from about 1 to about 100 times, to produce multiplealternating layers (or bilayers) of hematin and the polyelectrolyte onthe substrate. For a positively-charged substrate, the order of dippinginto hematin and a polyelectrolyte is reversed.

[0054] Polymerizations catalyzed by assembled hematins of the presentinvention can be carried out, for example, in a buffered solution,ranging from about pH 1 to about pH 12, at about 0° C. to about 50° C.An aromatic monomer and a template are added to the buffered solution,such that the ratio of aromatic monomer to template repeat unit is about5 to 1 to about 1 to 5. The concentration of aromatic monomer is about0.01 M to about 1 M. A quantity of assembled hematin, including about 2to about 100 bilayers of hematin and polyelectrolyte, is added to thesolution. A solution of a peroxide, in an amount sufficient topolymerize the aromatic monomer, is added dropwise over about 5 minutesto about 200 minutes. The reaction is maintained for about 1 hour toabout 200 hours. The progress of the reaction can be monitoredspectrophotometrically.

[0055] A peroxide, as used in the present invention, is an organic orinorganic compound that includes a —O— O— bond, such as ROOR, where R isas defined above. Preferably, one R is hydrogen, to give ROOH. Even morepreferably, the peroxide is hydrogen peroxide, HOOH.

[0056] Suitable substrates for assembled hematin are any solids that canmaintain an electrical charge. Examples of substrates include glasses(e.g., pyrex and glass slides), plastics (e.g., poly(vinyl chloride) andpoly(ethylene)), ceramics, metals, and the like. Preferred substratesare glass slides, which have been hydrophilized with an aqueous alkalisolution, such as Chem-solv, under ultrasonication.

[0057] The present invention will now be further described by thefollowing non-limiting examples.

EXAMPLE 1

[0058] Synthesis of PEG-Hematin Complex

[0059] The PEG-hematin complex was obtained through the coupling ofpolyethylene glycol (PEG) chains to a hematin molecule through esterlinkages as shown in FIG. 1. The PEG-hematin complex was prepared by theaddition of a mole equivalent of PEG (19 mg) to hematin (200 mg) in thepresence of activators N,N′-carbonyldiimidazole (0.05 g) and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.047 g) in DMF. The mixturewas allowed to stir for 48 hours then was quenched by the addition of alarge volume of deionized water. The unreacted reagents were removed byextraction with ethyl acetate. The water layer was subsequentlylyophilized to yield PEG-hematin as a reddish-brown solid.

[0060] The complex was characterized using NMR and FTIR spectroscopy.The average extent of modification of the acidic groups of hematin wasdetermined using UV-vis spectroscopy. The Uv-vis spectra of thePEG-hematin exhibited a decrease in the Soret band (420 nm), a porphyrincentered π−π* transition, in comparison to hematin, which was used tocalculate the amount of hematin present in the sample. However, theenergy and spectral bandwidths of PEG-hematin were indistinguishablefrom hematin, which indicated that the modification of hematin by poly(ethylene glycol) does not affect the heme structure. Based on thisassumption, the average concentration of hematin in the PEG-hematinsample was subsequently determined to be 67% by weight.

[0061] An FTIR spectrum of PEG-hematin indicated the presence of anester functionality by the appearance of a doublet at 1646 and 1651 cm⁻¹(similar to diethyl phthalate) accompanied by the complete disappearanceof the peak at 1712 cm⁻¹ for the acid carbonyl of hematin (FIG. 2). Thestrong peak at 1100 cm⁻¹ corresponded to the ether linkage of the glycolmoiety.

[0062] An ¹H NMR spectrum of PEG-hematin in DMF-d₇ shows thedisappearance of the peak at 10.2 ppm, which was assigned to thecarboxylic proton of hematin (FIG. 3a). This clearly indicated that thecarboxylic acid hydroxyl moiety was transformed into an ester. The largebroad peak at 3.8 ppm was assigned to the poly (ethylene glycol)protons. However the spectra could not be well resolved in the region of2-4 ppm due to the interference of the peaks assigned to the residualprotons in deuterated DMF. In order to get a better resolution of thespectrum, the solvent system was changed to deuterated water. Thespectrum in D₂O could not be used to distinguish the absence of thecarboxylic acid proton due to proton exchange with D₂O. However,comparison of the spectrum of PEG-hematin and the spectrum ofpoly(ethylene glycol), in D₂O showed the changes in the position of thePEG peaks of PEG-hematin in comparison to PEG alone. It was found thatPEG exhibited a major peak at 3.8 ppm, which was assigned to the bulk ofthe polymer chains, while the adjoining peaks (triplets) were assignedto the end groups of the polymer. When a PEG-hematin derivative wasformed, the peak at 4.0 ppm shifted upfield and merged into the mainpeak. This was accompanied by considerable broadening and a shift of thepeak at 3.8 ppm to 3.6 ppm (FIG. 3b). It was postulated that methyleneprotons α to the hydroxy group of PEG, on being attached by an esterlinkage to hematin, shifted upfield while methylene protons β to thehydroxy groups of PEG were affected by the inhomogeneous paramagneticenvironment, leading to broadening. These observed changes stronglyindicated the formation of an ester bond between PEG and hematin.

[0063] The activity of the PEG-hematin was assessed through theoxidation of pyrogallol (0.5%) to purpurogallin in 14 mM potassiumphosphate buffer in the presence of 0.027% (w/w) hydrogen peroxide.Interestingly, the activity of the PEG-hematin was found to beapproximately 30-fold higher as compared to native hematin at a pH 4.0(FIG. 4). Without being bound to any particular theory, it is believedthat the activity of hematin is dependent on its solubility. Thus, theenhanced activity of the PEG-hematin is attributed to its enhancedsolubility.

EXAMPLE 2

[0064] Synthesis of Polyaniline

[0065] The polymerization of aniline was carried out in 0.1 M sodiumphosphate buffer (10 mL) maintained at pH 1. To this buffer solution theaniline monomer was added. The catalyst, PEG-hematin (60 μg), was addedonly just prior to the addition of hydrogen peroxide. The polymerizationwas initiated by the incremental addition of a stoichiometric amount ofhydrogen peroxide, with respect to aniline. 0.3% H₂O₂ (w/v) was usedwith constant stirring and the progress of the reaction was monitoredspectroscopically (FIG. 5). Typically, all reaction systems were leftstirred until completion of polymerization followed by precipitation ofthe polyaniline. The Pani synthesized was filtered off and thoroughlywashed with acetone few times followed by drying in a vacuum oven. Theconductivity of the Pani pellet was found to be of the order of 0.2S/cm.

[0066] This reaction thus proved the versatility and ability of thePEG-Hematin for the synthesis of stable conducting polyaniline even inthe absence of template. The polyaniline formed in this case was againredox reversible as proved by cyclic voltammetry studies.

EXAMPLE 3

[0067] Synthesis of Sodium Poly (sodium-4-styrenesulfonate)-PolyanilineComplex

[0068] The polymerization of aniline was carried out in 0.1 M sodiumphosphate buffer over a range of pH conditions from pH 1-4. A 17 mMsolution of sodium polystyrene sulfonate (SPS) template in phosphatebuffer (100 mM) was prepared to which the aniline monomer was added in a1:1 molar ratio of aniline to sodium styrene sulfonate monomer. Thecatalyst, PEG-hematin (5 mg), was added just prior to the addition ofhydrogen peroxide. The polymerization was initiated by the incrementaladdition of a stoichiometric amount of hydrogen peroxide (relative toaniline). In all cases, 0.3% H₂O₂ (w/v) was used with constant stirring,and the progress of the reaction was monitored spectroscopically. Oncompletion of polymerization, the solution was transferred to individualregenerated natural cellulose membrane bags (molecular weight cut-off10,000 D) and were dialyzed against 5000 mL of acidified deionized watermaintained at pH 4.0 to remove unreacted monomers and oligomers. Thesolid SPS-polyaniline complex was obtained by evaporation of thedeionized water followed by drying in a vacuum oven.

[0069] It was observed that the solution slowly turned dark greenindicating the formation of the doped emeraldine salt form of conductingpolyaniline (polyaniline is hereinafter referred to as “Pani”). TheUV-vis absorption spectra of the Pani/SPS complex formed at differenttime intervals over a time period of 2 hours at pH 4.0 after initiationof polymerization reaction is shown in FIG. 6. The Uv-vis spectra showedthe presence of polaron absorption bands at 400 nm and 800-1200 nm whichwas consistent with the formation of the conducting form of polyaniline.This polymerization was also carried out at different pH values rangingfrom pH 1.0 to pH 4.0 as shown in FIG. 7. The formation of polyanilinewas observed in all cases, thus demonstrating the stability androbustness of the PEG-hematin in comparison to hematin (insoluble at lowpH) or horseradish peroxidase, HRP (denatured at low pH). Also thepolyaniline formation reaction catalyzed by PEG-hematin was found to becomplete with greater than 90% yield within a few hours, while theunmodified hematin showed little or no reactivity within the same timeperiod under these acidic conditions.

[0070] The redox tunability of the polyaniline formed was furtherdemonstrated by dedoping the emeraldine salt form of Pani at high pH andthen redoping with acid. With increasing pH (dedoping) on titration with1 N NaOH, the polaron bands at 400 nm and 800 nm were found to diminish,while a new band at 600 nm began to emerge due to the exciton transitionof the quinoid ring giving rise to a blue solution indicating that thePani has been fully dedoped to the base form. On titrating the solutionback with 1 N HCl (redoping), a reversible color change was observed andthe spectra is shown in FIG. 8. Furthermore, an isosbestic point at 710nm was also observed, which was indicative of the changes in thepolyaniline oxidation state. This behavior was similar to thepolyaniline synthesized chemically or enzymatically with HRP andconfirmed the formation of the conducting polyaniline emeraldine saltform (electroactive form) catalyzed by PEG-hematin.

[0071] The conductivity of the emeraldine salt form of polyanilinesynthesized at pH less than 4 was found to be about 10⁻³ S/cm.

[0072] Furthermore, cyclic voltammetry studies were carried out todetermine the electrochemical nature of polyaniline synthesized by thePEG-hematin catalysis. The cyclic voltammogram of a cast film of anSPS-Pani complex (FIG. 9) showed two sets of peaks indicating tworeversible redox cycles at a scan rate of 100 mV/s over a potentialwindow of −0.2-1.2 V.

EXAMPLE 4

[0073] Synthesis of Lignosulfonate-Pani Complex

[0074] 5.2 mg of a lignin sulfonate polyelectrolyte complex wasdissolved in 10 mL of sodium monophosphate buffer (0.1 M) maintained atpH 4.0. This was followed by the addition of 18 μL of aniline, acatalytic amount of PEG-Hematin and a amount of hydrogen peroxide (0.3%)stoichiometric with aniline. The reaction mixture was allowed to stiruntil precipitation of the polyelectrolyte-Pani complex ceased. Thereaction was also carried out in solutions having pHs ranging from pH1-4 (FIG. 10). The precipitated lignin sulfonate-Pani complex obtainedwas washed several times with acidified acetone to remove the unreactedmonomer and finally washed with acidified deionized water, filteredunder suction through a polycarbonate filter and dried in a vacuum ovento yield lignin sulfonate-polyaniline complex.

[0075] When the polymerization was conducted at pH 3.0, there was a peakof low intensity at 767 nm for the emeraldine form of polyaniline, whichwas completely absent during polymerization at pH 4.0. The extendedabsorption until 1200 nm indicated the formation of the extendedconjugation of the polyaniline backbone. Thus the synthesis of Panicomplexed with a natural polymer further widens the scope ofapplications to other natural polyelectrolytes to form versatile,environmentally benign conducting polymers.

EXAMPLE 5

[0076] Synthesis of DNA-Pani Complex The polymerization of aniline inthe presence of Calf Thymus DNA was carried out in sterile 10 mMphosphate buffer. A 1.0 mM calf thymus DNA solution was prepared bydissolving the required amount of DNA in 10 mL of sterilized sodiumphosphate buffer maintained at pH 4. The concentration of DNA wasdetermined by the UV absorbance at 258 nm. To this DNA solution, 4.5 μl(5 mM) of aniline was added. The pH of the solution was again checkedand adjusted to 4.3, and 5 mg of PEG-Hematin were added. To thisreaction mixture, a solution of hydrogen peroxide (0.3% solution, 4.5μl, 5 mM) was added drop-wise, to initiate the polymerization and thereaction of aniline was followed using UV-Vis spectroscopy and circulardichroism polarimetry.

[0077] When the aniline monomer was added to a DNA solution at pH 4.3,the electrostatic interaction between the protonated aniline monomersand the phosphate groups in the DNA caused the monomers to closelyassociate with the DNA. The association of the protonated anilinemonomer on the DNA template facilitated a predominantlypara-directedcoupling and inhibited parasitic branching during the polymerization.The high proton concentration around the phosphate groups also provideda unique local lower pH environment that permitted the polymerization ofaniline at a higher pH than that necessary with conventional chemicalpolymerization of aniline. The polymerization was catalyzed byPEG-hematin and initiated by hydrogen peroxide. However, as thepolymerization proceeded over a period of time and a critical chainlength was attained, the DNA-Pani complex precipitated out of solution.It was concluded that the complex remained soluble as long as there wereenough phosphate groups on the DNA available for solvation. As thepolymerization proceeded, the preferred molecular interaction betweenthe charged aniline groups and the phosphate groups of DNA caused thegrowing chain to occupy a majority of these sites leading to the saltingout of the DNA-Pani complex. The polymerization reaction was followedusing UV-vis spectroscopy and circular dichroism polarimetry. The UV-visspectra of the DNA-Pani complex recorded after initiation of thepolymerization are shown in FIG. 11. The UV-vis absorbance spectrashowed a peak around 260 nm emerging from the absorption of the basepairs of DNA along with polaron absorption bands at 420 nm and 750 nm,indicating the formation of the conducting emeraldine salt form ofpolyaniline.

[0078] The bases in the nucleic acid have a plane of symmetry and thusare not intrinsically optically active. However, the deoxyribose sugaris asymmetric and since the bases are attached to the anomeric carbon ofthese sugars, the sugar can induce a circular dichroism in theabsorption bands of the bases. These bands may be observed either forthe intensely electronically allowed π−π* transitions, or for the weaklyallowed n−π* transitions because these transitions are magneticallyallowed. Also the π electron systems of the bases make them hydrophobic,so the bases tend to stack in hydrogen-bonding solvents to minimize theπ-electron surface area exposed to the solvent. The hydrophobic planesand hydrophilic edges as well as charge-charge interactions cause thebases to stack and the polymer to adopt a helical structure.Preferential handedness is induced in these helical structures by theintrinsically asymmetric sugars, giving the DNA polymer a whole superasymmetry. The electronic transitions of these chromophoric bases are inclose proximity and can thus interact to give well-defined CD spectra.The CD spectrum of the DNA-Pani complex showed a reduction in theintensity of the peak at 275 nm (FIG. 12). This change indicated apolymorphic transition in DNA causing the DNA to change from a looselywound form to the over-wound form. The appearance of a positive peak at450 nm indicated that the helical polyelectrolyte DNA template induces amacroscopic order in the Pani that is formed. This result proves theextensive versatility of the PEG-Hematin catalyst with a variety oftemplates including delicate biomacromolecules in providing the optimalcatalytic activity for polymerization.

EXAMPLE 6

[0079] Synthesis of Poly(2-methoxy-5-methylaniline)-SPS Complex

[0080] The polymerization of 2-methoxy-5-methylaniline (2M5M) wascarried out in 0.1 M sodium phosphate buffer of pH 4.0. A 17 mM SPStemplate solution, as measured from the concentration of sodium styrenesulfonate monomers, in phosphate buffer (10 mL) was prepared to which2M5M (24 mg) was added in the desired (1:1, 2M5M:SPS) molar ratio. Thepolymerization was initiated after addition of 5 mg of PEG-Hematin, bythe incremental addition of an amount of peroxide (0.3% w/v)stoichiometric with 2M5M, with constant stirring. The progress of thereaction was monitored spectroscopically. After the reaction wascomplete, the solution was dialyzed to remove the unreacted monomers,followed by evaporation to yield a SPS-poly(2M5M) complex.

[0081] The UV-vis absorption spectra of the poly (2M5M)/SPS complexformed is shown in FIG. 14. The spectra again showed the presence of apolaron band at 425 nm and extended conjugation in the longer wavelengthrange indicating the linear conducting form of polyaniline. This polymeralso showed reversible redox tunability similar to that observed for theSPS-polyaniline complex formed in Example 2. The SPS-poly(2M5M) formedcould also be reversibly de-doped on titrating with 1 N NaOH andre-doped by back titrating with 1N HCl.

EXAMPLE 7

[0082] Synthesis of Sodium Dodecylbenzenesulfonic Acid-Pani Complex

[0083] Polymerization of aniline was carried out in 0.1 M sodiumphosphate buffer at pH 4. A 17 mM solution of dodecylbenzenesulfonicacid (DBSA) in phosphate buffer (100 mM) was prepared to which theaniline monomer was added in the desired (1:1, Aniline:DBSA) molarratio. The catalyst, PEG-Hematin (5 mg), was added just prior to theaddition of hydrogen peroxide. The polymerization was initiated by theincremental addition of an amount of hydrogen peroxide stoichiometric toaniline. In all cases, 0.3% H₂O₂ (w/v) was used with constant stirring.The progress of the reaction was monitored spectroscopically.

EXAMPLE 8

[0084] Synthesis of SPS-Polyphenol Complex

[0085] A polymerization reaction was carried out in 10 mL of aqueousphosphate buffer (100 mM). The pH of the reaction media for the phenolpolymerization was maintained at pH 7.0 and equimolar concentrations (17mM) of SPS, with respect to the concentration of the repeat units, andphenol monomer were added to the buffered solution, followed by 10 mg ofthe PEG-hematin. The reaction was initiated by addition of astoichiometric, with respect to phenol, amount of H₂O₂ (30% w/v) in onelot to facilitate the formation of high molecular weight polyphenol. Thereaction was monitored spectroscopically. A control experiment was alsocarried out simultaneously in the absence of catalyst. The finalproducts were dialyzed using Centricon concentrators (10,000 Mw cut off,Amicon Inc., Beverly, Mass.) to remove unreacted monomers. The sampleswere then dried under vacuum at 50° C. and used for further analysis.The yield was calculated to be typically 95% or higher.

[0086] The PEG-hematin complex was also found to catalyze thepolymerization of phenol at pH 7.0 more efficiently than that comparedto the native hematin and peroxidase (FIG. 15). The large broadabsorption tail in the region from 300-700 nm confirmed the presence ofextended conjugation and indicated formation of polyphenol byPEG-hematin reaction. In comparison, the absorption of thehematin-catalyzed reaction is relatively weak. Thus, modification of thehematin with PEG was observed to significantly improve the reactivity tosuit the desired reaction conditions leading to the formation ofpolyphenol.

EXAMPLE 9

[0087] Preparation of Assembled Hematin

[0088] Glass slides (25 by 75 mm) were hydrophilized with 1% Chem-solvsolution in deionized water under ultrasonication for use as substrates.This treatment generates negative charges on the surface of the slidesdue to partial hydrolysis. After 3 hr, the slides were ultrasonicatedtwice in deionized water for 30 min before use.

[0089] The electrostatic layer-by-layer deposition process was carriedout in two steps. Poly(diallyldimethylammonium chloride) (PDAC) (10 mM)and hematin (3 mM) solutions were prepared over a pH range from 5 to 11.In the first step, hydrophilized glass slides were immersed in PDACsolution for 10 min at room temperature and washed with deionized waterfor 5 min. After the deposition and washing steps, the slides were driedwith a stream of nitrogen. In the second step, the substrates with asingle layer of PDAC were immersed into the hematin solution for 10 minand subsequently washed with deionized water and dried with a stream ofnitrogen to produce an assembled hematin, having a bilayer film ofPDAC/hematin. This dipping procedure was iterated to build up multilayerfilms.

EXAMPLE 10

[0090] Synthesis of Pani-SPS Complex Using Assembled Hematin

[0091] Polymerization of aniline was carried out at room temperature ina 40 mL, 0.1M phosphoric acid buffer solution, which contained a 1:1molar ratio of SPS (MW 1,000,000; moles correspond to quantity ofmonomers units) to aniline 0.167 g (0.81 mmol). SPS was added first tothe buffered solution, followed by an addition of 2.1 mL of anilinestock solution (0.036 mL aniline to 1 mL buffer at pH 1.4) with constantstirring. A seventeen bilayer Hematin/PDAC assembly was immersed in thesolution. To initiate aniline polymerization, 11 mL of 0.25% H₂O₂ wasadded dropwise, incrementally, over 30 min. The reaction was maintainedfor 24 h, and carried out at different pH values (1.0, 2.0, 3.0). Therate of assembled hematin catalyzed polymerization was monitored by aPerkin-Elmer Lamda-9-UV-vis spectrophotometer at room temperature.

[0092] Equivalents

[0093] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of polymerizing an aromatic monomer,comprising combining an aromatic monomer with a hematin catalyst,wherein the hematin catalyst has been derivatized with one or morenon-proteinaceous amphipathic groups.
 2. The method of claim 1, furthercomprising combining a peroxide initiator with the aromatic monomer andthe derivatized hematin.
 3. The method of claim 2, further comprising atemplate, wherein the aromatic monomer aligns along said template andpolymerizes to form a complex comprising the polymerized aromaticmonomer and the template.
 4. The method of claim 3, wherein the templateis a polyelectrolyte.
 5. The method of claim 4, wherein thepolyelectrolyte is polyanionic.
 6. The method of claim 5, wherein thepolyanionic polyelectrolyte is poly(styrene sulfonic acid) or a saltthereof.
 7. The method of claim 3, wherein the template is opticallyactive.
 8. The method of claim 7, wherein the optically active templateis an oligonucleotide or a polynucleic acid or a salt thereof.
 9. Themethod of claim 8, wherein the polynucleic acid is 2′-deoxyribonucleicacid or a salt thereof.
 10. The method of claim 5, wherein the templateis lignin sulfonic acid or a salt thereof.
 11. The method of claim 5,wherein the template is dodecylbenzene sulfonic acid or a salt thereof.12. The method of claim 3, wherein the aromatic monomer is a substitutedor unsubstituted aromatic compound.
 13. The method of claim 12, whereinthe aromatic compound is an aniline.
 14. The method of claim 13, whereinthe aniline is 2-methoxy-5-methylaniline.
 15. The method of claim 12,wherein the aromatic compound is a phenol.
 16. The method of claim 13,wherein the complex formed is a water-soluble complex of a polyanilineand the template.
 17. The method of claim 16, wherein the polyaniline isof the electrically-conducting emeraldine salt form.
 18. The method ofclaim 15, wherein the complex formed is a water-soluble complex ofpolyphenol and the template.
 19. The method of claim 7, wherein thepolymerized aromatic monomer complexed to the template has amacro-asymmetry.